Increased long-term immunity to Bacillus anthracis protective antigen in mice immunized with a CIA06B-adjuvanted anthrax vaccine

Anthrax is an acute infectious disease caused by Bacillus anthracis. We previously reported that the adjuvant CIA06B, which consists of TLR4 agonist CIA05 and aluminum hydroxide (alum), enhanced the immune response to anthrax protective antigen (PA) in mice. This study was carried out to determine whether CIA06B can enhance long-term immune responses to PA in mice. BALB/c mice were immunized intramuscularly three times at 2-week intervals with recombinant PA alone or PA combined with alum or CIA06B. At 8 and 24 weeks post-immunization, the immunological responses including serum anti-PA IgG antibody titer, toxin-neutralizing antibody titer, splenic cytokine secretion and the frequency of PA-specific memory B cells were assessed. Compared with mice injected with PA alone or PA plus alum, mice injected with PA plus CIA06B had higher titers of serum anti-PA IgG antibodies, and higher frequencies of PA-specific memory B cells and interferon-γ secreting cells. Furthermore, anti-PA antibodies induced by CIA06B were more effective in neutralizing anthrax toxin. These results demonstrated that CIA06B is capable of providing long-term immunity when used as an adjuvant in a PA-based anthrax vaccine.     

A combination of the TLR4 agonist CIA05 and alum promotes the immune responses to Bacillus anthracis protective antigen in mice

Anthrax is an infectious disease caused by Bacillus anthracis. The currently licensed human anthrax vaccines contain protective antigen (PA) as a major protective component and alum as an adjuvant. In this study, we investigated whether CIA05, a TLR4 agonist, is able to promote the immune response to an anthrax vaccine adjuvanted with alum. BALB/c mice were immunized intraperitoneally three times at 2-week intervals with a recombinant B. anthracis PA alone or in combination with CIA05 in the absence or presence of alum, and immune responses were determined 2 or 3 weeks after the third immunization. The results showed that the combination of CIA05 and alum significantly increased both serum anti-PA IgG antibody and toxin-neutralizing antibody titers, and the adjuvant effects were greater when lower antigen doses were used for immunization. Both CIA05 and alum stimulated PA-specific splenocyte secretion of interleukin (IL)-4, IL-5, and IL-6. A combination of the two yielded synergistic effects on IL-4 secretion, but CIA05 tended to repress IL-5 and IL-6 secretions induced by alum. Co-administration of CIA05 and alum also increased GL7 expression in B220(+)CD24(+) splenic cells, indicating the ability to activate B cells. These data suggest that CIA05, combined with alum, could be used to achieve higher immune responses to PA, leading to the development of an effective anthrax vaccine.     

Strong Mucosal and Systemic Immunities Induced by Nasal Immunization with Anthrax Protective Antigen Protein Incorporated in Liposome–Protamine–DNA Particles

Purpose The very lengthy and complicated dosing schedule of the current anthrax vaccine adsorbed, which was licensed in the USA for the prevention of cutaneous anthrax infection, calls for the development of an efficacious and easily administrable vaccine to prevent against the most lethal form of anthrax infection, the inhalation anthrax. We propose to develop a nasal anthrax vaccine using anthrax protective antigen (PA) protein carried by liposome–protamine–DNA (LPD) particles. Methods PA was incorporated in LPD particles and nasally dosed to mice. The resulting PA-specific immune response and lethal toxin neutralization activity were measured. Results Mice nasally immunized with PA incorporated into LPD particles developed both systemic and mucosal anti-PA responses. The anti-PA immunities induced included the production of anti-PA antibodies (IgG and IgM in the serum and IgA in nasal and lung mucosal secretions) and the proliferation of splenocytes after in vitro stimulation. The anti-PA IgG subtype induced was mainly IgG1. Finally, anthrax lethal toxin neutralization activity was detected both in the serum and in the mucosal secretions. Conclusions The anti-PA immune response induced by nasal PA incorporated in LPD was comparable to that induced by nasal PA adjuvanted with cholera toxin or subcutaneously injected PA adjuvanted with aluminum hydroxide.     

Evaluation of the immune response induced by a nasal anthrax vaccine based on the protective antigen protein in anaesthetized and non-anaesthetized mice

To better protect against inhalational anthrax infection, a nasal anthrax vaccine based on the protective antigen (PA) protein of Bacillus anthracis could be an attractive alternative to the current Anthrax-Vaccine-Adsorbed (AVA), which was licensed for cutaneous anthrax prevention. Previously, we have demonstrated that an anti-PA immune response comparable with that in mice subcutaneously immunized with PA protein adjuvanted with aluminium hydroxide was induced in both the systemic compartment and the mucosal secretions of the nose and lung of anaesthetized mice when they were nasally immunized with PA protein incorporated into previously reported LPD (Liposome-Protamine-DNA) particles. In this study, we evaluated the anti-PA immune response induced by the nasal PA/LPD particles in non-anaesthetized mice and compared it with that in anaesthetized mice. Our data showed that the anti-PA antibody response and the anthrax lethal toxin-neutralization activity induced by the nasal PA/LPD in non-anaesthetized mice was relatively weaker than that in anaesthetized mice. However, the splenocytes isolated from the nasally immunized mice, anaesthetized and non-anaesthetized, proliferated comparably after in-vitro re-stimulation. By evaluating the uptake of fluorescence-labelled LPD particles by phagocytes in the nasal and broncho-alveolar lavages of mice after the nasal administration, we concluded that the relatively weaker anti-PA immune response in the non-anaesthetized mice might be partially attributed to the reduced retention of the PA/LPD particles in the nasal cavity of the non-anaesthetized mice. Data collected in this study are expected to be useful for future anthrax nasal vaccine studies when mice are used as a model.     

Cellular and humoral adjuvant activity of lectins isolated from Korean mistletoe (Viscum album colaratum)

The adjuvant effect of lectins (KML-C) isolated from Korean mistletoe (Viscum album coloratum) on induction of humoral and cellular immune responses against keyhole limpet hemocyanine (KLH) was examined. When mice were immunized subcutaneously (s.c.) with KLH (20 micrograms/mouse) admixed with or without 50 ng/mouse of KML-C (KLH + KML-C), mice immunized with KLH + KML-C showed significantly higher antibody titers against KLH than those immunized with KLH alone, showing the highest titer 5 weeks after immunization. Furthermore, boost immunization with KLH + KML-C at 2-week interval elicited much higher activity than single immunization to enhance antibody responses against KLH. The assay for determining isotypes of antibodies revealed that KML-C augmented KLH-specific antibody titers of IgG1, IgG2a and IgG2b. The culture supernatants obtained from the splenocytes of mice treated with KLH + KML-C also showed a higher level of both KLH-specific Th-1 (IL-2 and IFN-gamma) and Th-2 type cytokine (IL-4). In an in vitro analysis of T lymphocyte proliferation to KLH on week 4, the splenocytes of mice treated with KLH + KML-C showed a significantly higher proliferating activity than those treated with KLH alone. In addition, mice immunized twice with KLH + KML-C and followed by intrafootpad (i.f.) injection of KLH (50 micrograms/site) 14 weeks after the primary immunization induced a higher delayed-type hypersensitivity (DTH) reaction than mice treated with KLH alone. These results suggest that KML-C is a potent immunoadjuvant to enhance cellular and humoral immune responses.     

Suppressive effects of Hochu-ekki-to, a traditional Chinese medicine, on IgE production and histamine release in mice immunized with ovalbumin

We examined the effects of Bu-Zhong-Yi-Qi-Tang (Japanese name: Hochu-ekki-to, HET), a traditional Chinese medicine, on IgE production and histamine release in mice immunized intraperitoneally with a mixture of ovalbumin (OA) and aluminum hydroxide (alum adjuvant). Three groups of mice were orally administered 0, 1.7 or 17 mg of HET on day 13 after the first immunization with a mixture of 1 microg OA and 1 mg alum adjuvant. They were again immunized with the same dose of OA plus alum adjuvant on day 14. The immunological changes in mice treated with OA alone or OA plus HET were examined, and the following findings were obtained. In the HET-treated mice, the elevation of anti-OA IgE in serum, and histamine release from basophils in blood, were significantly suppressed. A significant suppression of interleukin-4 (IL-4) secretion and proliferation of splenic lymphocytes in primary culture was also observed. A tendency to suppress the elevation of anti-OA IgG1 in serum and interleukin-2 (IL-2) secretion from splenic lymphocytes was observed in the HET-treated mice. These findings suggest that oral administration of HET suppresses IgE antibody production and histamine release in type I allergic reaction in mice immunized with OA plus alum adjuvant; this shows the efficacy of HET in treating type I allergic diseases, such as asthma.     

Topical immunization onto mouse skin using a microemulsion incorporated with an anthrax protective antigen protein-encoding plasmid

The current anthrax vaccine in the U.S., the anthrax vaccine adsorbed, has several serious drawbacks, most notably the very lengthy and complicated dosing schedule. Thus, there is a critical need to develop an alternative anthrax vaccine with a simplified immunization schedule. To address this need, we evaluated the feasibility of topically priming or boosting onto the skin using an anthrax protective antigen (PA) protein-encoding DNA vaccine. To this end, we have shown that topical immunization of mice onto their skin with a perflubron-based microemulsion incorporated with a PA63-encoding plasmid, pGPA, led to significant PA-specific antibody responses, which have anthrax lethal toxin-neutralization activity. Moreover, topical boosting of mice primed with PA protein with the pGPA-incorporated, perflubron-based microemulsion significantly enhanced the anti-PA antibody responses induced. This topical anthrax DNA vaccine has the potential to be combined with a vaccine, such as the current AVA, to produce a simplified and more convenient dosing schedule.     

Nasal Immunization with Anthrax Protective Antigen Protein Adjuvanted with Polyriboinosinic–Polyribocytidylic Acid Induced Strong Mucosal and Systemic Immunities

Purpose The current anthrax vaccine adsorbed (AVA) was originally licensed for the prevention of cutaneous anthrax infection. It has many drawbacks, including the requirement for multiple injections and subsequent annual boosters. Thus, an easily administrable and efficacious anthrax vaccine is needed to prevent the most lethal form of anthrax infection, inhalation anthrax. We propose to develop a nasal anthrax vaccine using anthrax protective antigen (PA) protein as the antigen and synthetic double-stranded RNA in the form of polyriboinosinic–polyribocytidylic acid (pI:C) as an adjuvant. Methods Mice were nasally immunized with recombinant PA admixed with pI:C. The resulting PA-specific antibody responses and the lethal toxin neutralization activity were measured. Moreover, the effect of pI:C on dendritic cells (DCs) was evaluated both in vivo and in vitro. Results Mice nasally immunized with rPA adjuvanted with pI:C developed strong systemic and mucosal anti-PA responses with lethal toxin neutralization activity. These immune responses compared favorably to that induced by nasal immunization with rPA adjuvanted with cholera toxin. Poly(I:C) enhanced the proportion of DCs in local draining lymph nodes and stimulated DC maturation. Conclusions This pI:C-adjuvanted rPA vaccine has the potential to be developed into an efficacious nasal anthrax vaccine.     

Structure-function relationship of the saponins from the roots of Platycodon grandiflorum for hemolytic and adjuvant activity

To assess the contribution of the aglycone and sugar chain to the biological activity of saponins from Platycodon grandiflorum, seven structurally consecutive saponins, platycodin D (PD), D2 (PD2), D3 (PD3), platycoside A (PA), E (PE), deapioplatycoside E (DPE), and polygalacin D2 (PGD) were compared for their hemolytic activities and adjuvant potentials on the immune responses to Newcastle disease virus-based recombinant avian influenza vaccine (rL-H5) in mice. Among seven compounds, the order of the hemolytic activity was PGD ≈ PD > PD2 > PA > PD3 > PE > DPE. PD, PD2, PA, and PGD significantly not only promoted concanavalin A (Con A)-, lipopolysaccharide (LPS)- and antigen-induced splenocyte proliferation, but enhanced the NK cell activity in mice immunized with rL-H5. PD and PD2 increased the antigen specific IgG, IgG1, IgG2a, and IgG2b antibody titers, while PA and PGD only induce the IgG and IgG1 antibody responses in the immunized mice. However, the other three saponins were not observed for adjuvant activity. The results suggested that the sugar chains attached to C-3, the glycidic moiety at C-28 of aglycone, as well as aglycone affect their biological activities. Interestingly, their hemolytic and adjuvant activities increased with the retention time by reverse phase HPLC analysis. The retention time may be useful for primary estimation of fundamental adjuvanticity of saponin with the same aglycone.     

Immunological adjuvant effect of Boswellia serrata (BOS 2000) on specific antibody and cellular response to ovalbumin in mice

In this study, the biopolymeric fraction BOS 2000 from Boswellia serrata was evaluated for its potential ability as adjuvants on the immune responses to ovalbumin (OVA) in mice. Balb/c mice were immunized subcutaneously with OVA 100 μg alone or with OVA 100 μg dissolved in saline containing alum (200 μg) or BOS 2000 (10, 20, 40 and 80 μg) on Days 1 and 15. Two weeks later, OVA specific antibodies in serum; concanavalin A (Con A), OVA stimulated splenocyte proliferation, CD4/CD8/CD80/CD86 analysis in spleen cells and its estimation of cytokines (IL-2 and IFN gamma) from cell culture supernatant were measured. OVA specific IgG, IgG1 and IgG2a antibody levels in serum were significantly enhanced by BOS 2000 (80 μg) compared with OVA control group. Moreover, the adjuvant effect of BOS 2000 (80 μg) on the OVA-specific IgG, IgG1, and IgG2a antibody responses to OVA in mice were more significant than those of alum. BOS 2000 significantly enhanced the Con A and OVA induced splenocyte proliferation in the OVA immunized mice especially at a dose of 80 μg (p<0.001). However, no significant differences were observed among the OVA group and OVA/alum group. At a dose of 80 μg (p<0.001), there was a significant increase in the CD4/CD8 and CD80/CD86 analysis in spleen cells and cytokine (IL-2 and IFN-gamma) profile in the spleen cell culture supernatant was observed. In conclusion, BOS 2000 seems to be a promising balanced Th1 and Th2 directing immunological adjuvants which can enhance the immunogenicity of vaccine.     

Oligodeoxynucleotides containing synthetic immunostimulatory motifs augment potent Th1 immune responses to HBsAg in mice

Toll-like receptor 9 (TLR9) modulators have potent Th1-adjuvant activity. We recently reported the development of immunomodulatory oligonucleotides (IMOs) containing novel structures (immunomers) and synthetic immunostimulatory CpR (R=2'-deoxy-7-deazguanosine) or R'pG (R'=1-(2'-deoxy-beta-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs. IMOs activate TLR9 pathways, resulting in cytokine secretion profiles different from those induced by CpG DNA. In the present study we evaluated the adjuvant activity of IMOs containing CpG, CpR, or R'pG motifs in combination with hepatitis B surface antigen (HBsAg) in a mouse model. Mice immunized with HBsAg plus IMO produced higher levels of IgG2a and lower levels of IgG1 than did mice immunized with HBsAg alone or with alum. High IgG2a responses were found at week 4 and remained high until 14 weeks after immunization. Adoptive transfer of splenocytes from HBsAg/IMO-immunized mice to na?ve mice resulted in strong IgG2a production in response to antigen boost. Splenocytes of mice immunized with HBsAg/IMO produced high levels of IFN-gamma, but not Th2 cytokines IL-4 and IL-5, in antigen-recall experiments in vitro. The use of IMOs as adjuvants to HBsAg resulted in the production of strong anti-HBsAg antibodies at antigen doses as low as 0.2 microg. These data demonstrate that IMOs enhance the immunogenicity of HBsAg through potent Th1 immune responses, which may allow lower doses of antigen in vaccination.     

Recombinant viral-like particles of parvovirus B19 as antigen carriers of anthrax protective antigen

Viral-like particles (VLPs) of parvovirus B19 were employed as an antigen carrier to present antigenic determinants of Bacillus anthracis. The small-loop peptide and the full-length domain 4 of protective antigen (PA) were chosen as immunogens for presentation on the VLP-capsid surface and subsequent immunization of BALB/c mice. The recombinant VLPs induced anti-PA IgG titers of up to 2.5 x 10(4). Neutralization assays showed that the recombinant VLPs elicited neutralizing anti-PA antibody titers of up to 1:400 and showed potential for the prevention of lethal toxin-induced mortality of mouse-macrophage cells (RAW264.7). In postimmune sera, no anti-PA titers were detected against synthetic small-loop peptide. Recombinant VLPs demonstrated the capacity to retain the immunogenicity of the displayed microbial PA-epitopes and elicited robust levels of anti-PA antibody titers. These findings suggest that the recombinant VLPs of parvovirus B19 have potential as an additional tool in the development of sub-unit vaccines.     

Negatively charged liposomes show potent adjuvant activity when simply admixed with protein antigens

Liposomes have been investigated extensively as a vaccine delivery system. Herein the adjuvant activities of liposomes with different net surface charges (neutral, positive, or negative) were evaluated when admixed with protein antigens, ovalbumin (OVA, pI = 4.7), Bacillus anthracis protective antigen protein (PA, pI = 5.6), or cationized OVA (cOVA). Mice immunized subcutaneously with OVA admixed with different liposomes generated different antibody responses. Interestingly, OVA admixed with net negatively charged liposomes prepared with DOPA was as immunogenic as OVA admixed with positively charged liposomes prepared with DOTAP. Immunization of mice with the anthrax PA protein admixed with the net negatively charged DOPA liposomes also induced a strong and functional anti-PA antibody response. When the cationized OVA was used as a model antigen, liposomes with net neutral, negative, or positive charges showed comparable adjuvant activities. Immunization of mice with the OVA admixed with DOPA liposomes also induced OVA-specific CD8(+) cytotoxic T lymphocyte responses and significantly delayed the growth of OVA-expressing B16-OVA tumors in mice. However, not all net negatively charged liposomes showed a strong adjuvant activity. The adjuvant activity of the negatively charged liposomes may be related to the liposome's ability (i) to upregulate the expression of molecules related to the activation and maturation of antigen-presenting cells and (ii) to slightly facilitate the uptake of the antigens by antigen-presenting cells. Simply admixing certain negatively charged liposomes with certain protein antigens of interest may represent a novel platform for vaccine development.     

Enhanced immune response with a combination of alum and biodegradable nanoparticles containing tetanus toxoid

The purpose of the study was to determine the synergistic adjuvant effect of sustained release biodegradable nanoparticles in combination with alum. Nanoparticles containing tetanus toxoid (TT) were formulated using a biodegradable polymer, polylactic polyglycolic acid co-polymer (50:50, molecular weight 100 000). The immunization studies were carried out subcutaneously in rats. The results were expressed as mean serum anti-TT IgG levels. The nanoparticles demonstrated a TT loading of 4% w/w with mean particle diameter of 238 #45 31 nm. The TT encapsulated in nanoparticles was released slowly under in vitro conditions, with 67.5% cumulative release occurring in 20 days. A single injection of TT-nanoparticles (TT dose=10 Lf) mixed with TT-Alum (TT dose =5 Lf) induced a four-fold greater mean serum anti-TT IgG response than a single injection of TT nanoparticles alone (TT dose=15 Lf) (2235 #45 310 vs. 539 #45 49 #119 g/ml, mean #45 sem, p => 0.001). In addition, the mean immune response induced with the single injection of combination of nanoparticles and alum was comparable to the two injections of TT-alum alone (5 Lf each dose) given at 3 week intervals IgG = 1998 #45 333 #119 g/ ml). Furthermore, the combination induced a peak immune response (IgG = 4215 #45 546 #119 g/ml) as early as the first time point at 3 weeks post-immunization. In the case of a TT-alum alone, the animals showed a weaker immune response at 3 weeks and required a second dose of TT-alum to enhance the antibody response. The data thus suggest that the combination of TTnanoparticles and TT-alum acts as a much better adjuvant than nanoparticles or alum alone. A rapid induction of immune response is useful to curb the spread of communicable diseases in the case of an outbreak.     

Enhanced immune response with a combination of alum and biodegradable nanoparticles containing tetanus toxoid.

The purpose of the study was to determine the synergistic adjuvant effect of sustained release biodegradable nanoparticles in combination with alum. Nanoparticles containing tetanus toxoid (TT) were formulated using a biodegradable polymer, polylactic polyglycolic acid co-polymer (50:50, molecular weight 100000). The immunization studies were carried out subcutaneously in rats. The results were expressed as mean serum anti-TT IgG levels. I'he nanoparticles demonstrated a TT loading of 4% w/w with mean particle diameter of 238 +/- 31 nm. The TT encapsulated in nanoparticles was released slowly under in vitro conditions, with 67.5% cumulative release occurring in 20 days. A single injection of TT-nanoparticles (TT dose = 10 Lf) mixed with TT-Alum (TT dose= 5 Lf) induced a four-fold greater mean serum anti-TT IgG response than a single injection of TT nanoparticles alone (TT dose=15Lf) (2235 +/- 310 vs. 539 +/- 49 microg/ml, mean +/- sem, p = >0.001). In addition, the mean immune response induced with the single injection of combination of nanoparticles and alum was comparable to the two injections of TT-alum alone (5 Lf each dose) given at 3 week intervals IgG = 1998 +/- 333 microg/ ml). Furthermore, the combination induced a peak immune response (IgG = 4215 +/- 546 microg/ml) as early as the first time point at 3 weeks post-immunization. In the case of a TT-alum alone, the animals showed a weaker immune response at 3 weeks and required a second dose of TT-alum to enhance the antibody response. The data thus suggest that the combination of TT-nanoparticles and TT-alum acts as a much better adjuvant than nanoparticles or alum alone. A rapid induction of immune response is useful to curb the spread of communicable diseases in the case of an outbreak.     

Generation of mouse polyclonal and human monoclonal antibodies against Bacillus anthracis toxin

High titer antisera against the protective antigen (PA) from Bacillus anthracis were generated immunizing Balb/c mice two times intraperitoneally with PA in combination with lipopeptide adjuvant P3CSK4. The sera were able to protect the mouse macrophage cell line J774A.1 from an anthrax toxin challenge. We also tested the blood of anthrax vaccine-immunized persons for PA- and lethal factor (LF)-specific antibodies. An increased titer was found after three immunizations, and the sera were also able to protect the mouse macrophage cell line from a toxin challenge. For the preparation of human monoclonal antibodies, we used peripheral blood lymphocytes. After in vitro stimulation using PA or synthetic peptides derived from PA, B lymphocytes were immortalized by PEG fusion with the human mouse heteromyeloma cell line CB-F7. We obtained several clones producing high amounts of PA-specific immunoglobulin (Ig).     

Influence of particle size, antigen load, dose and additional adjuvant on the immune response from antigen loaded PLA microparticles

Polylactide (PLA) polymer particles entrapping tetanus toxoid (TT) were evaluated in terms of particle size, antigen load, dose and additional adjuvant for achieving high and sustained anti-TT antibody titer from single point intramuscular immunization. Admixture of polymer entrapped TT and alum improved the immune response in comparison to particle-based immunization. High and long lasting antibody titer was achieved upon immunization with 2-8 microm size particles. Microparticles within the size range 50-150 microm elicited very low serum antibody response. Immunization with very small particles (<2 microm) and with intermediate size range particles (10-70 microm) elicited comparable antibody response from single point immunization but lower in comparison to that achieved while immunizing with 2-8 microm size particles. Potentiation of antibody response on immunization of admixture of microparticles and alum was also dependent on particle size. These results indicate the need of optimal particle sizes in micron ranges for improved humoral response from single point immunization. Increasing antigen load on polymer particles was found to have positive influence on generation of antibody titers from particle based immunization. Maximum peak antibody titer of approximately 300 microg/mL was achieved on day 50 upon immunization with particles having highest load of antigen (94 microg/mg of polymer). Increase in dose of polymer entrapped antigen resulted in concomitant increase in peak antibody titers indicating the importance of antigen stability, particle size and load on generating reproducible immune response. Optimization of particle size, antigen load, dose and use of additional adjuvant resulted in high and sustained anti-TT antibody titers over a period of more than 250 days from single point immunization. Serum anti-TT antibody titers from single point immunization of admixrure of PLA particles and alum was comparable with immunization from two divided doses of alum adsorbed TT.     

Evaluation of clinical and serological findings for diagnosis of cutaneous anthrax infection after an outbreak

Purpose: Anthrax, caused by the bacterium Bacillus anthracis, is one of the oldest documented infectious diseases in both livestock and humans. We aimed to evaluate clinical findings and risk factors of patients with cutaneous anthrax infection and report anti-lethal factor (LF) IgG and anti-protective antigen (PA) IgG titers in the serologic diagnosis of disease.

Methods: In this study, serum samples of 18 cutaneous anthrax patients were collected and anti-LF IgG and anti-PA IgG titers were measured by enzyme-linked immunosorbent assay (ELISA).

Results: Twelve (67%) males and 6 (33%) females, with a mean age of 36.06?±?16.58 years were included in the study. Risk factors identified in the patient population studied were slaughtering (28%), flaying (56%), chopping meat (67%), burying diseased animal corpses (17%) and milking (6%) livestock. Black eschar formation (94%), pruritus (78%) and painful lymphadenopathy (61%) were first three common clinical signs and symptoms, respectively. Fourteen (78%) patients produced a positive IgG response against PA, 11 (61%) patients produced against LF. Three (17%) patients had no response to either antigen.

Conclusions: A detailed history of contact with sick animals or animal products along with clinical findings should be taken at the first step for the diagnosis of cutaneous anthrax infection. Serologic detection of anti-LF IgG and anti-PA IgG with ELISA may be useful auxillary method for establishing the diagnosis.     

细粒棘球蚴延伸因子-1重组蛋白对小鼠的免疫保护性研究

目的:探讨细粒棘球蚴(Eg)延伸因子(Eg.EF-1)重组蛋白对小鼠的免疫保护性及其作为候选疫苗的潜在价值。方法:ICR小鼠随机分为免疫组和佐剂对照组,每隔2周皮下免疫1次,在第3次免疫后2周,用Eg原头节进行攻击感染,感染后20周剖杀小鼠,检获Eg包囊,计算免疫保护力,并用酶联免疫吸附法测定血清中免疫球蛋白(Ig)G及其亚型和IgE水平。结果:与佐剂对照组比较,免疫组小鼠的免疫保护力为85.6%,血清IgG、IgG1、IgG2a水平显著性升高(P<0.05),IgG2b水平轻微降低(P>0.05),IgG3和IgE水平无明显变化。结论:rEg.EF-1重组蛋白能诱导小鼠产生一定的免疫保护力,是潜在的疫苗候选抗原分子。